基于人类免疫缺陷病毒-1（HIV-1）的慢病毒载体是一种用于基因转移研究的载体。慢病毒载体可以将外源基因或外源的shRNA有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。慢病毒载体具有能感染非分裂期细胞、容纳外源性目的基因片段大、稳定性强、免疫原性小等特点。大量研究表明，相对其他病毒载体，慢病毒感染效率高，更容易感染一些较难感染的组织和细胞，其效率一般可以达到30%-95%以上。在感染能力方面，慢病毒载体可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞。对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，使用慢病毒载体能大大提高目的基因或目的shRNA的转导效率，且目的基因或目的shRNA整合到宿主细胞基因组的几率大大增加，能够比较方便快捷地实现目的基因或目的shRNA的长期、稳定表达。在体外实验及体内实验的研究中，慢病毒己经成为表达外源基因或外源shRNA的常用载体形式之一，并且正在获得越来越广泛的应用。
病毒颗粒的限制性摄取往往会降低基因或shRNA表达的成功率，这要求实验者使用更高滴度的病毒颗粒以达到理想的基因表达效率。然而，增加病毒数量以促进表达可能会导致许多细胞类型的毒副作用，带来不必要的昂贵成本。
阿拉丁为此研发了独特的Viral Transfection Enhancer慢病毒转导增强剂，专为提高慢病毒基因转导效率而设计。Viral Transfection Enhancer是一种高效、无细胞毒性的慢病毒化学转导增强剂。它是一种非离子型、非受体依赖性的表面活性剂，通过与细胞膜相互作用，降低细胞膜粘稠度，提高脂质交换和跨膜转运，促进慢病毒与细胞膜的融合，从而提高病毒转导效率。同时，Viral Transfection Enhancer介导的转导增强，不依赖特异的细胞表面受体，在转导过程中避免了细胞膜的去极化和损伤，不影响细胞活力。研究证实，Viral Transfection Enhancer在不影响转导细胞存活、增殖及分化能力的前提下，提高了小鼠T细胞、HSCs和人HSCs的慢病毒转导效率。
Viral Transfection Enhancer作为一种普遍作用的（受体非依赖型）佐剂，可广泛应用于多种难转导的细胞类型，包括CD34+造血干细胞（HSCs），间充质干细胞（MSCs），神经干细胞，原代T细胞，NK细胞和成纤维细胞等，对难转导的小鼠T细胞亦有效。
产品特点：
⁕ 即用型试剂，无需进行试剂配置，简单方便。
⁕ 无菌制剂，可直接加入细胞使用。
⁕ 增强慢病毒转导效率可高达 90%，有利于取得理想的基因表达效率。
⁕ 在转导过程中避免了细胞膜的去极化和损伤，不影响转导细胞存活、增殖及分化能力。
实验步骤：
1. 包装慢病毒并测定慢病毒滴度，通过病毒滴度和MOI计算目的细胞感染所需的病毒量。
2. 实验前一天，将生长状态良好的目的细胞消化计数后稀释至1-3×10⁵/mL，加入24孔板，500μL/孔（0.5-1.5x10⁵个/孔），放入37℃，5% CO₂培养箱中培养。保证第二天进行病毒感染时细胞汇合度约为30%-50%。
⁕ 悬浮细胞不需要提前一天接种，实验前将细胞计数接种后，直接加入病毒混合液即可。
3.配制慢病毒+Viral Transfection Enhancer+培养基混合液。Viral Transfection Enhancer推荐使用的稀释比例为1:500-1:100。
⁕ 相比于添加polybrene所产生的细胞毒性来说，Viral Transfection Enhancer对细胞的毒性极低，几乎对细胞生长无任何损伤，因此，针对不能使用polybrene的细胞，Viral Transfection Enhancer是首选的病毒感染增强剂。
4.吸去24孔板中目的细胞的培养基，加入3中配制的慢病毒混合液，放入37℃，5% CO₂ 培养箱中继续培养。
⁕ 感染悬浮细胞或半悬浮细胞，则需采用平角离心转染法，即将适量的病毒液加入细胞培养板后，封好口，放入平角离心机中，800xg离心30分钟（因细胞而异），然后放入培养箱中继续培养。
5.病毒感染16 h后更换为新鲜培养基继续培养。
6. 病毒感染48 h后，可加入筛选培养基进行稳定细胞株筛选，或采用荧光显微镜、流式细胞术、western blot、qPCR等方法检测目的基因表达情况。
适用范围：
Viral Transfection Enhancer 作为一种普遍作用的（受体非依赖型）佐剂，可广泛应用于多种难转导的细胞类型，在不影响转导细胞存活、增殖及分化能力的前提下，能够显著提高慢病毒转导效率。
注意事项：
1. 相比于添加 polybrene 所产生的细胞毒性来说，Viral Transfection Enhancer 对细胞的毒性极低，几乎对细胞生长无任何损伤，因此，针对不能使用 polybrene 的细胞，Viral Transfection Enhancer 是首选的病毒感染增强剂。
2. 为了您的健康安全，请规范操作，穿戴实验服与手套开展实验。
3. 所有慢病毒操作均需在 BSL2 级生物安全柜中进行。
4. 本产品仅供科研使用，请勿用于临床诊断及治疗。
Lentiviral vectors based on Human Immunodeficiency Virus-1 (HIV-1) are widely used as tools for gene transfer research. These vectors can efficiently integrate exogenous genes or short hairpin RNA (shRNA) into the host cell genome, enabling long-term stable expression of the target sequence. Lentiviral vectors possess distinct advantages, including the ability to infect non-dividing cells, large capacity for exogenous target gene fragments, high stability, and low immunogenicity. Numerous studies have demonstrated that compared to other viral vectors, lentiviruses exhibit higher infection efficiency—they can more easily infect refractory tissues and cells, with an efficiency typically ranging from 30% to over 95%. In terms of infectivity, lentiviral vectors can effectively transduce a variety of cell types, such as neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells. For hard-to-transfect cells (e.g., primary cells, stem cells, and undifferentiated cells), lentiviral vectors significantly improve the transduction efficiency of target genes or shRNA. Additionally, they increase the likelihood of integrating the target gene/shRNA into the host cell genome, facilitating convenient and rapid long-term stable expression of the target sequence. Lentiviral vectors have become one of the most commonly used carriers for expressing exogenous genes or shRNA in both in vitro and in vivo studies, and their applications are expanding increasingly.
However, the restricted uptake of viral particles often reduces the success rate of gene or shRNA expression. This forces researchers to use higher-titer viral particles to achieve optimal gene expression efficiency. Nevertheless, increasing viral dosage to enhance expression may cause toxic side effects in many cell types and incur unnecessary high costs.
To address this challenge, Aladdin has developed a unique Viral Transfection Enhancer—a lentiviral transduction enhancer specifically designed to improve lentiviral gene transduction efficiency. As a highly efficient, non-cytotoxic chemical enhancer for lentiviral transduction, it is a non-ionic, receptor-independent surfactant. By interacting with the cell membrane, it reduces membrane viscosity, enhances lipid exchange and transmembrane transport, and promotes the fusion of lentiviruses with the cell membrane—ultimately boosting viral transduction efficiency. Notably, the transduction enhancement mediated by Viral Transfection Enhancer is independent of specific cell surface receptors. During the transduction process, it avoids cell membrane depolarization and damage, thus not affecting cell viability. Research has confirmed that Viral Transfection Enhancer improves lentiviral transduction efficiency in mouse T cells, mouse hematopoietic stem cells (HSCs), and human HSCs without compromising the survival, proliferation, or differentiation ability of transduced cells.
As a universally acting (receptor-independent) adjuvant, Viral Transfection Enhancer is broadly applicable to various hard-to-transduce cell types, including CD34+ hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), neural stem cells, primary T cells, natural killer (NK) cells, and fibroblasts. It is also effective for refractory mouse T cells.
Product Features:
⁕ Ready-to-use reagent, no need for reagent preparation, simple and convenient.
⁕ Sterile preparation, can be directly added to cells for use.
⁕ Can enhance lentiviral transduction efficiency by up to 90%, facilitating the achievement of ideal gene expression efficiency.
⁕ Avoids depolarization and damage of cell membranes during transduction, without affecting the survival, proliferation, or differentiation ability of transduced cells.
Experimental Procedures:
1. Package the lentivirus and determine its titer. Calculate the amount of virus required for target cell infection based on the virus titer and MOI (Multiplicity of Infection).
2. One day before the experiment: Digest the target cells in good growth state, count them, and dilute to a concentration of 1–3×10⁵ cells/mL. Seed the cells into a 24-well plate at a volume of 500 μL per well (corresponding to 0.5–1.5×10⁵ cells per well). Incubate the plate in a 37°C, 5% CO₂ incubator. Ensure that the cell confluency reaches approximately 30%–50% on the day of viral infection.
⁕ Suspension cells do not need to be seeded one day in advance. On the day of the experiment, count and seed the cells, then directly add the virus mixture.
3. Prepare the lentivirus + Viral Transfection Enhancer + medium mixture. The recommended dilution ratio for Viral Transfection Enhancer is 1:500 to 1:100.
⁕ Compared with the cytotoxicity caused by polybrene addition, Viral Transfection Enhancer exhibits extremely low cytotoxicity and causes almost no damage to cell growth. Therefore, for cells that cannot tolerate polybrene, Viral Transfection Enhancer is the preferred viral infection enhancer.
4. Aspirate the medium from the target cells in the 24-well plate, add the lentivirus mixture prepared in Step 3, and return the plate to the 37°C, 5% CO₂ incubator for continued culture.
⁕ For infecting suspension cells or semi-suspension cells, use the flat-angle centrifugation transfection method: Add an appropriate amount of virus solution to the cell culture plate, seal the plate tightly, place it in a flat-angle centrifuge, centrifuge at 800×g for 30 minutes (centrifugation parameters may vary by cell type), then transfer the plate to the incubator for continued culture.
5. Sixteen hours after viral infection, replace the medium with fresh medium and continue culturing.
6. Forty-eight hours after viral infection: Either add selection medium to screen for stable cell lines, or detect the expression of the target gene using methods such as fluorescence microscopy, flow cytometry, Western blot, or qPCR (quantitative Polymerase Chain Reaction).
Scope of Application:
As a universally acting (receptor-independent) adjuvant, Viral Transfection Enhancer can be widely applied to various hard-to-transduce cell types. It significantly improves lentiviral transduction efficiency without affecting the survival, proliferation, or differentiation ability of transduced cells.
Precautions:
1. Compared with the cytotoxicity caused by adding polybrene, Viral Transfection Enhancer has extremely low toxicity to cells and causes almost no damage to cell growth. Therefore, for cells that cannot use polybrene, Viral Transfection Enhancer is the preferred viral infection enhancer.
2. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves when conducting experiments.
3. All lentivirus-related operations must be performed in a Biosafety Level 2 (BSL2) biological safety cabinet.
4. This product is for research use only and shall not be used for clinical diagnosis or treatment.
ZJ25F1230162
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